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United States Patent |
6,103,238
|
Essex
,   et al.
|
August 15, 2000
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Selectively deglycosylated human immunodeficiency virus type 1 envelope
vaccines
Abstract
Selective deglycosylation of HIV-1 envelope proteins enhances their ability
to elicit a protective immune response in people. Glycosylation can reduce
or prevent immunological recognition of envelope protein domains.
Selective deglycosylation exposes these domains and improves the
opportunity for a protective immune response. Deglycosylation which
produces substantial conformational changes (as determined by loss of
infectivity) should be avoided. Recombinant HIV-1 envelope glycoproteins
are generated which have primary amino acid sequence mutation(s) in
consensus sequence(s) for N-linked glycosylation (sugar attachment), so as
to prevent glycosylation at that site(s). The position of such genetic
deglycosylation is important and should be between the C terminus of gp120
and the Cys at the N-terminal side of the cysteine loop containing the
hyper-variable region 3 (V3) (this Cys is generally positioned about at
residue 296, counting from the N-terminus of gp120). The mutant
glycoprotein should be deglycosylated such that the total molecular mass
of the mutant gp120 component is less than 90% (more preferably less than
75%) of the corresponding fully glycosylated wild type gp120 component to
maximize a useful immune response.
Inventors:
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Essex; Myron E. (Sharon, MA);
Lee; Tun-Hou (Newton, MA);
Lee; Woan-Ruoh (Brookline, MA);
Lee; Chun-Nan (Brookline, MA)
|
Assignee:
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President and Fellows of Harvard College (Cambridge, MA)
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Appl. No.:
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850770 |
Filed:
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March 13, 1992 |
Current U.S. Class: |
424/188.1; 424/130.1; 424/139.1; 424/141.1; 424/142.1; 424/147.1; 424/148.1; 424/208.1; 530/387.5; 530/387.9; 530/388.35; 530/395 |
Intern'l Class: |
A61K 039/21; A61K 039/395; A61K 039/42; A61K 039/40 |
Field of Search: |
424/89,130.1,188.1,133.1,208.1,141.1,142.1,147.1,148.1
530/395,387,398,388.1,387.1,387.9,388.38
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References Cited
Other References
Bolmstedt, et al, J of Gen Virology, 72:1269-1277, 1991.
Woan-Ruoh, et al, Final Program and Abstracts, 6th International Conference
on Aids, San Francisco, CA 3:S.A.8, 1990.
Feizi, et al, Glycobiology 1:17-23, 1990.
Cohen, et al, J of Acquired Immune Deficiency Syndromes, 3:11-18, 1990.
Syu, et al, PNAS, 87:3695-3699, 1990.
Aldovini, et al, J of Virology, 64:1920-1926, 1990.
Leonard, et al, The J of Biol Chem, 265:10373-10382, 1990.
Kozarsky, et al, J of Acquired Immune Deficiency Syndromes, 2:163-169,
1989.
Cohen, et al, Nature, 334:532-534, 1988.
Willey, et al, J of Virology, 62:139-147, 1988.
Chou, et al, The J of Infectious Disease, 157:805-811, 1988.
Geyer, et al, The J of Bio Chem, 263:11760-11767, 1988.
Curran, et al, Science, 239:610-616, 1988.
Mizuochi, et al, Biochem J, 254:599-603, 1988.
Lasky, et al, Cell, 50:975-985, 1987.
Matthews, et al, PNAS, 84:5424-5428, 1987.
Pyle, et al, Aids Research and Human Retroviruses, 3:387-400, 1987.
Lasky, et al, Science, 233:209-212, 1986.
McDougal, et al, Science, 231:382-385, 1986.
Cullen, et al, Cell, 46:973-982, 1986.
Robey, et al, PNAS, 83:7023-7027, 1986.
Veronese, et al, Science, 229:1402-1404, 1985.
Robey, et al, Science, 228:593-595, 1985.
Klatzmann, et al, Nature, 321:767-768, 1984.
Dalgleish, et al, Nature, 312:763-767, 1984.
Allan, et al, Science 228:1091-1094, 1985.
Botarelli et al., "N-glycosylation of HIV-gp120 may constrain recognition
by T lymphocytes", J. Immunol., 147(9):3128-32 (1991).
Lee et al., "Non-random Distribution of GP 120 N-linked Glycosylation Sites
Critical for HIV-1 Infectivity", AIDS Research and Human Retroviruses,
8(5):917 (1992).
Lee et al., "Nonrandom distribution of gp120 N-linked Glycosylation Sites
Important for Infectivity of Human Immunodeficiency Virus Type 1", PNAS
USA, 89(6):2213-17 (1992).
Dirckx et al.; Mutation of conserved n-glycosylation . . . ; Vir. Res.;
vol. 18(1); pp. 9-20, Dec. 1990.
Bolmstedt et al.; Effects of mutations in glycosylation sites . . . ; J.
Gen. Vir.; vol. 72; pp. 1269-1277, Jun. 1991.
Lee, et al, 1990, "Role of N-linked Oligosaccharides of HIV-1 . . . "
Abstract No. S.A.8, 6.sup.th International Conference on AIDS, Jun. 24,
1990.
Davis, et al., 1990, "Glycosylation Governs the Binding of . . . " J. of
General Virology 71:2889-2898.
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Primary Examiner: Stucker; Jeffrey
Assistant Examiner: Nelson; Bret
Attorney, Agent or Firm: Fish & Richardson, P.C.
Goverment Interests
The invention was supported by the U.S. Government which has certain rights
in the invention.
Claims
What is claimed is:
1. A composition comprising a mutant recombinant human immunodeficiency
virus type 1 (HIV-1) envelope glycoprotein which is mutated in its primary
amino acid sequence with respect to a wild type HIV-1 envelope
glycoprotein, said mutant glycoprotein including two or more N-linked
carbohydrate consensus amino acid sequence mutations so as to effect
partial deglycosylation, said mutation being positioned between the C
terminus of gp120 and the Cys at the N-terminal side of the gp120 cysteine
loop containing the third hypervariable sequence (V3), said Cys being
approximately at amino acid position 296, said mutant glycoprotein being
sufficiently deglycosylated such that the total molecular mass of the
mutant gp120 component is less than 75% of the corresponding fully
glycosylated wild type gp120 component, said mutant glycoprotein being
effective, when present as a component of a complete HIV virion, to enable
viral infectivity.
2. The mutant glycoprotein composition of claim 1, wherein said virus is
human immunodeficiency virus type 1, strain selected from the group
consisting of MN, HXB2, IIIB, LAI, NL43, MFA, BRVA, SC, JH3, ALAI, BALI,
JRCSF, OYI, SF2, NY5CG, SF162, JFL, CDC4, SF33, AN, ADA, WMJ2, RF, ELI,
Z2Z6, NDK, JY1, MAL, U455, and Z321.
3. The mutant glycoprotein composition of claim 1, wherein said
glycoprotein is gp160.
4. The mutant glycoprotein composition of claim 1, wherein said
glycoprotein is gp120.
5. The mutant glycoprotein composition of claim 1, wherein said primary
amino acid sequence is mutated such that one or more consensus N-linked
glycosylation sequence mutation is a substitution of Asn, Ser, or Thr with
a different amino acid.
6. The mutant glycoprotein composition of claim 1 wherein there are
deglycosylations at multiple N-linked glycosylation attachment sites in
the region between the C terminus of gp120 and the Cys on the N-terminal
side of the cysteine loop containing hypervariable region 4 (V4).
7. The mutant glycoprotein composition of claim 1 in which at least one of
the N-linked glycosylation sequences corresponding to positions 289 and
356 are not mutated.
8. The mutant glycoprotein of claim 1 in which at least one of the N-linked
glycosylation sequences corresponding to the following position is
deglycosylated: 386, 392, 397, 406 and 463.
9. A method of producing antibodies comprising:
(a) administering to a mammal a mutant envelope protein, said protein being
mutated in its primary amino acid sequence with respect to a wild type
HIV-1 envelope glycoprotein, said mutant glycoprotein including two or
more N-linked carbohydrate consensus amino acid sequence mutations so as
to effect partial deglycosylation, said mutations being positioned between
the C terminus of gp120 and the Cys at the N-terminal side of the gp120
cysteine loop containing the third hypervariable sequence (V3), said Cys
being approximately at amino acid position 296, said mutant glycoprotein
being sufficiently deglycosylated such that the total molecular mass of
the mutant gp120 component is less than 75% of the corresponding fully
glycosylated wild type gp120 component, said mutant glycoprotein being
effective, when present as a component of a complete HIV virion, to enable
viral infectivity; and
(b) recovering said antibodies.
10. The antibodies of claim 9 wherein said antibodies are monoclonal
antibodies.
Description
BACKGROUND OF THE INVENTION
The field of the invention is human immunodeficiency virus vaccines and
immunotherapeutics.
Human immunodeficiency virus is the etiological agent of acquired immune
deficiency syndrome (AIDS). The env gene of HIV encodes a 160 kD
glycoprotein that is subsequently cleaved into two smaller species, an
extracellular (or surface) protein gp120 and a transmembrane protein gp41
(Allan et al., 1985, Science 228:1091; Di'Marzo-Veronese et al., 1985,
Science 229:1402). Gp120 is noncovalently linked to gp41 (Allan et al.,
1985, Science 228:1091; Chou et al., 1988, J. Infect. Dis. 157:805;
Di'Marzo-Veronese et al., 1985, Science 229:1402; Lasky et al., 1987, Cell
50:975).
Among the various HIV isolates, some sequences are highly conserved and
some are variable. Two characteristics of the env glycoprotein are
conservation of cysteine residues and of a relatively large number of
N-linked carbohydrate sites in HIV-1 isolates. Similar secondary and
tertiary structures for the env glycoprotein have been suggested based on
the similarity of the sequences of HIV.
The env glycoprotein is heavily glycosylated. The unmodified polypeptide
backbone of gp120 (about 480 amino acids) weighs about 55 kD. About one
half of the molecular weight of gp120 can be accounted for by attached
carbohydrates (Allan et al., 1985, Science 228:1091; Geyer et al., 1988,
J. Biol. Chem. 263:11760; Matthews et al., 1987, Proc. Natl. Acad. Sci.
USA 84:5424; Mizuochi et al., 1988, Biochem J. 254:599; Robey et al.,
1985, Science 228:593). Although gp41 is also a glycoprotein, it is not as
heavily glycosylated as gp120 (Di'Marzo-Veronese et al., 1985, Science
229:1402). The oligosaccharides of the gp120/41 complex are generally
N-linked with no detectable O-linked sugar residues present (Kozarsky et
al., 1989, J. AIDS 2:163; Leonard et al., 1990, J. Biol. Chem. 265:10373).
The consensus sequence of the site for N-linked carbohydrate attachment is
Asn-X-Ser/Thr, where X is any amino acid except Pro and Asp. HIV-1
molecular clones contain an average of 23-24 potential N-linked
carbohydrate attachment sites on gp120 and about 4-7 on gp41. The
consensus sites on gp120 are generally glycosylated when the env protein
is expressed in chinese hamster ovary (CHO) cells (Leonard et al., 1990,
J. Biol. Chem. 265:10373).
CD4 is the host cell receptor for HIV (Dalgleish et al., 1984, Nature
312:763; Klatzmann et al., 1984, Nature 312:767; McDougal et al., 1986,
Science 231:382). The CD4-binding domain of HIV has been mapped to the
C-terminal region of gp120 (Kowalski et al., 1987, Science 231:1351; Lasky
et al., 1987, Cell 50:975), although it is reported that sequences in the
N-terminal region of gp120 may also be involved (Syu et al., 1990, Proc.
Natl. Acad. Sci. USA 87:3695).
Vaccines and immunotherapeutics comprising native gp120 and gp160 have been
proposed.
SUMMARY OF THE INVENTION
We have discovered that selectively deglycosylated HIV-1 envelope proteins
retain their ability to support viral infectivity, implying that they
generally retain the native envelope conformation. We also noted that the
envelope protein of the related simian virus for African green monkeys
(SIV.sub.AGM), which is not pathogenic to its natural host, has fewer
N-linked glycosylation sites, particularly in the C-terminal portion of
the surface envelope protein analogous to gp120. Without wishing to bind
ourselves to a specific detailed molecular explanation, we propose that a
selectively deglycosylated HIV-1 envelope protein is more effective in
eliciting a protective immune response in people. Glycosylation serves to
reduce or prevent immunological recognition of envelope protein domains.
Selective deglycosylation enables an immune response to these domains and
improves the opportunity for a protective immune response. Deglycosylation
which produces substantial conformational changes (as determined by loss
of infectivity) should be avoided.
We have further found that the invention can be achieved by generating
recombinant HIV-1 envelope glycoproteins which have primary amino acid
sequence mutation(s) in consensus sequence(s) for N-linked glycosylation
(sugar attachment), so as to prevent glycosylation at that site(s).
Moreover, we have found that the position of such genetic deglycosylation
is important. Preferably, the position of such genetic deglycosylation
should be between the C terminus of gp120 and the Cys at the N-terminal
side of the cysteine loop containing the hypervariable region 3 (V3) (this
Cys is generally positioned about at residue 296, counting from the
N-terminus of gp120). We have found that it is important to remove at
least a minimum amount of the total native gp120 carbohydrate in order to
maximize the opportunity for a useful immune response. Specifically, the
mutant glycoprotein should be deglycosylated such that the total molecular
mass of the mutant gp120 component is less than 90% (more preferably 75%)
of the corresponding fully glycosylated wild type gp120 component.
Another indicia of a suitable conformation for a desirable immune response
is infectivity--i.e., the mutant glycoprotein (when present as a component
of a complete HIV-1 virion) enables viral infectivity. By retaining viral
infectivity, we mean that when the envelope gene of HIV or an infectious
DNA clone is engineered to encode the mutations of the mutant envelope
glycoprotein, the virus retains infectivity.
By wild-type or native HIV-1 envelope glycoprotein we mean the envelope
glycoprotein encoded by a naturally occurring HIV-1 isolate. With respect
to designation of amino acid positions of the envelope glycoprotein such
as the Cys at the N-terminal side of the cyteine loop containing V3
(approximately amino acid position 296), it will be understood that
certain aspects of envelope structure are conserved throughout virtually
all HIV-1 strains, and those conserved structures can be used as
landmarks. For example cysteine cross-links form loops which contain
hypervariable regions having widely accepted designations.
By the term "recombinant glycoprotein" we mean a glycoprotein produced by
expression of a DNA sequence that does not occur in nature and which
results from human manipulations of DNA bases. The term envelope
glycoprotein means gp160, gp120, or other env-encoded peptides containing
at least the above-described C-terminal portion of gp120.
Accordingly, one aspect of the invention features compositions comprising
mutant selectively deglycosylated HIV-1 recombinant envelope glycoproteins
as described above. Other aspects of the invention feature vaccines (both
for protecting uninfected individuals and for treating infected
individuals) that comprise such mutant HIV-1 recombinant envelope
proteins. Still other aspects of the invention feature DNA encoding the
mutant HIV-1 recombinant envelope proteins (particularly in an expression
vector), recombinant cells comprising such DNA, and methods of making the
recombinant mutant envelope glycoproteins by expressing such DNA. Still
another aspect of the invention features antibodies raised, or
preferentially binding to, the mutant envelope glycoprotein.
In preferred embodiments, mutants of either gp120 or gp160 can be used.
Because the deglycosylation unmasks envelope regions which are generally
conserved, it is possible to use any of a wide range of HIV-1 strains or
isolates e.g., MN, HXB2, LAI, NL43, MFA, BRVA, SC, JH3, ALAI, BALI, JRCSF,
OYI, SF2, NY5CG, SF162, JFL, CDC4, SF33, AN, ADA, WMJ2, RF, ELI, Z2Z6,
NDK, JY1, MAL, U455, Z321. The preferred mutation at the consensus
N-linked glycosylation sequence is substitution of Asn, Ser or Thr with a
different amino acid (i.e., any amino acid other than the one occupying
the position in the wild type). Preferably, there are multiple
deglycosylations in the above described C-terminal region, particularly in
the region between the C terminus of gp120 and the Cys on the N-terminal
side of the cysteine loop containing hypervariable region 4 (V4). For
example, one or more of the positions 386, 392, 397, 406 or 463 may be
deglycosylated. We have found that in some cases the consensus sequence
closest to position 448 and/or position 392 may be mutated, together with
other C-terminal consensus sequence mutations. We have also found that it
is preferable to maintain glycosylation at the consensus sequence closest
to position 289. It may also be desirable in some constructions to
maintain glycosylation at position 356. For convenience the numbers given
above gp120 refer to amino acid residues of the HXB2 envelope protein.
Those skilled in the field will understand that conservation of envelope
features in other strains will permit the application of the invention to
the envelope proteins of those strains. For example, there is conservation
of cysteine cross-links that define loops with hypervariable regions.
Thus, the reference to positions 386, 392, 397, 406 and 463 can be
understood as a reference to the N-linked glycosylation sites positioned
between the C-terminus of gp120 and the Cys on the N-terminal side of the
cysteine loop containing hypervariable region 4 (V4). Similarly, the
reference to positions 289 and 356 can be applied to other strains with
reference to FIG. 1 and FIG. 2.
Other features and advantages of the invention will be apparent from the
following description of preferred embodiments and from the claims.
DETAILED DESCRIPTION
The drawings are first briefly described.
Drawings
FIG. 1 is a diagram depicting the conservation of N-linked glycosylation
sites in gp120 of selected HIV-1 isolates. Twenty-four consensus N-linked
glycosylation sites of HXB2 are shown by lines. The numbers above each
line indicate the amino acid positions in HXB2. The longer lines with an
asterisk symbol represent N-linked glycosylation sites not present in
HXB2.
FIG. 2 is a schematic drawing of gp120. Darkened lines represent the
hypervariable regions of the molecule which form 5 loops, designated V1-5,
via cysteine-cysteine disulfide bonds which are represented by the solid
lines connecting each end of a loop. The numbers represent the first amino
acid in each of the 24 potential N-linked glycosylation sites in the
molecule.
FIG. 3 is a schematic diagram of gp120 from HIV-1. The distribution and
amount of conservation of N-linked glycosylation sites is shown. Amino
acids are numbered from the N-terminus of the molecule to the C-terminus.
The numbers beneath the diagram denote the position of the first amino
acid in the consensus sequence of an N-linked glycosylation site. Sites
which are .gtoreq.90% conserved among HIV-1, HIV-2 and SIV isolates are
indicated by an arrow with a solid head and are numbered sequentially with
the prefix `a`. Sites which are at least 50% conserved are indicated by an
arrow with an open head and are numbered sequentially with the prefix `b`.
Other sites which are conserved at a level of less than 50% are indicated
by an arrow with a wavy tail.
FIG. 4 is a western blot demonstrating expression of gp160 and gp120 in
COS-1 cells transfected with wild type or mutant proviral DNA. Cell
lysates from transfected COS-1 cells were separated on 12%
SDS-polyacrylamide gels, transferred to nitrocellulose filters, and then
reacted with a reference sheep anti-gp120 serum. The wild type virus is
abbreviated WT and N-linked glycosylation mutants are indicated by numbers
representing their position in HXB2.
FIG. 5 is a graphical demonstration of infection of CD4-positive SupT1
cells by N-linked glycosylation mutants. Reverse transcriptase activity in
cultured supernatants of SupT1 cells infected by wild type (WT) virus and
by mutant viruses 141 or 197, was measured over a period of 28 days. The
growth kinetics of mutants 88, 160 and 276 were similar to those of mutant
141. The growth kinetics of mutant 262 was similar to those of mutant 197.
The growth kinetics of other first-site N-linked glycosylation mutants
were similar to those of wild type virus.
FIG. 6 is a western blot analysis of the envelope glycoproteins expressed
by wild type and mutant viruses. COS-7 cell lysates were prepared 48 hours
post-transfection and electrophoresed on 12% SDS-PAGE, transferred to
nitrocellulose, and reacted with sheep anti-gp120 antisera. (A) Mock, wild
type and C2, C3, C4, C5 and C6 mutants. (B) Mock, wild type, N2, N3, N4
and N5 mutants.
FIGS. 7A and B are graphical demonstrations of RT activity in SupT1 cells
infected with wild type and mutant viruses. (A) Mock, wild type and C2,
C3, C4, C5 and C6 mutants. (B) Mock, wild type and N2, N3, N4 and N5
mutants.
GENERATION OF MOLECULES USEFUL AS VACCINE CANDIDATES FOR HIV-1
As outlined above, proteins according to the invention are recombinant
human immunodeficiency virus envelope glycoproteins which are mutated with
respect to a wild type (native) human immunodeficiency virus glycoprotein
in the primary amino acid sequence to effect partial deglycosylation. The
genetic change should be introduced to positions in the C-terminal portion
of gp120 (between the C-terminus of gp120 and a specific cysteine which
forms the loop containing V3). Notwithstanding the mutation(s), the
conformation of the glycoprotein remains sufficiently intact to maintain
infectivity when present as a component of the virion. We propose that, in
individuals that are immunized with this molecule, an immune response will
be induced to reduce or block viral infectivity.
As illustrated by the studies described below, potential N-linked
glycosylation sites in gp120 can be systematically mutated, either singly
or in combination by site directed mutagenesis such that the consensus
glycosylation sequence is disrupted. Recombinant viruses are generated
containing gp120 genes that have such mutations. To determine whether the
conformation is retained in the mutated gp120, the infectivity of each
mutant virus is measured. Processing of gp160 to gp120 and gp41 may also
be assessed as a rough measure of retention of conformation and
infectivity.
In general there are more than 20 consensus N-linked glycosylation sites in
the gp120 coding sequence of HIV-1 isolates. For illustrative purposes, we
have shown the positions of these sites on gp120 in HXB2 and in other
strains of HIV-1 in FIG. 1. The relative positions of these sites on the
predicted structure of gp120 in HXB2 are shown in FIG. 2. A linear map of
the conserved N-linked glycosylation sites, their relative positions and
their level of conservation are presented in FIG. 3.
In FIG. 3, the following residue designations correspond to the arrows of
gp120:
______________________________________
a1 = 88 a5 = 241 a9 = 356
a2 = 136 a6 = 262 b9 = 386
b1 = 141 a7 = 276 b11 = 392
a3 = 156 b5 = 289 b12 = 397
b2 = 160 a8 = 295 b13 = 406
a4 = 186 b6 = 301 a10 = 448
b3 = 197 b7 = 332 a11 = 463
230 not b8 = 339
marked
b4 = 234
______________________________________
Sequence information for envelope proteins of other strains (e.g. the
strains listed above) are referenced in Myers et al. Human Retroviruses
and AIDS (1991): "A compilation and analysis for nucleic acid and amino
acid sequences" (Los Alamos National Laboratory, Los Alamos, N. Mex.),
which is hereby incorporated by reference.
The following studies are provided to illustrate (not to limit) the
invention, and particularly to illustrate methods for readily determining
the relative importance of each of the various HIV envelope N-linked
glycosylation sites and the effect of mutations to those sites and
combinations thereof.
Mutation of Potential N-Linked Glycosylation Sites and the Effect of these
Mutations on Envelope Glycoprotein Viral Infectivity
The molecular clone HXB2, which contains 24 N-linked glycosylation sites
was used as the template DNA for site-directed mutagenesis as follows.
Construction of Mutants
Oligonucleotide-directed mutagenesis was performed on a 2.7 Kb SalI-BamHI
fragment of HXB2 (Cohen et al., 1990, J. AIDS 13:11), which covers all 24
N-linked glycosylation sites of gp120, using the method of Kunkel (Cohen
et al., 1988, Nature 334:532). The oligonucleotide primers used for
mutagenesis were synthesized using standard cyanoethyl phosphoamidite
chemistry and are listed in Table I. Mutants were identified by the Sanger
chain-termination method (Cullen, 1986, Cell 46:973). The SalI-BamHI
fragment containing the desired mutation was excised from the replicative
form of each mutant and used to replace the 2.7 Kb SalI-BamHI fragment of
HXB2. All HXB2-derived N-linked glycosylation site mutants containing the
designated changes were further verified by DNA sequencing (Cullen, 1986,
Cell 46:973).
Western Blot Analysis of Envelope Proteins
Ten micrograms of wild type or mutant DNA was transfected into
3-5.times.10.sup.6 COS-1 cells using DEAE-dextran (Curran et al., 1988,
Science 239:610). Cells lysates were collected 48 hours after
transfection. Mock-transfected, wild type, and mutant transfected COS-1
cells were washed with phosphate-buffered saline (PBS) once and subjected
to centrifugation at 2500 rpms. Cell pellets were resuspended with 100 ml
RIPA lysis buffer (0.15 M NaCl/0.05 M Tris HCl pH 7.2, 1% Triton X-100, 1%
Sodium deoxycholate, 0.1% SDS) and spun down at 35,000 rpm (Ti70 rotor;
Beckman) at 4.degree. C. for 40 minutes. Ten microliters of cell lysates
were electrophoresed in 12% SDS-polyacrylamide gels. A reference HIV-1
positive serum at a 1:200 dilution and a sheep anti-gp120 (AIDS Research
Reference Reagent Program #288) at 1:2000 dilution were used for western
blots as described (Dalgleish et al., 1984, Nature 312:763).
Monitoring of Syncytium-formation and Viral Infectivity
The CD4 positive human T lymphoid cell line, SupT1, was grown and
maintained at 37.degree. C. in RPMI-1640 containing 10% heat-inactivated
fetal bovine serum and 1% penicillin-streptomycin. COS-1 cells were
propagated in Dulbecco's minimal eagle medium supplemented with 10%
heat-inactivated fetal bovine serum and 1% penicillin-streptomycin.
Cell-free supernatants were collected 48 hours after transfection.
Supernatants were filtered through 0.45 mm filters and assayed for
virion-associated reverse transcriptase (RT) activity. Equal amounts of
wild type and mutant virus, as measured by RT activity (100K cpm), was
used to infect 1.times.10.sup.6 SupT1 cells. One milliliter of the culture
medium was collected every three or four days and assayed for RT. Cultures
were monitored for 28 days.
Reverse Transcriptase Assay
One milliliter of culture medium was mixed with 0.5 ml 30% PEG and 0.4M
NaCl on ice for 2 hours and spun at 2500 rpm at 4.degree. C. for 30
minutes. The pellet was resuspended in 100 ml of RT buffer (0.5% Triton
X-100, 15 mM Tris pH 7.4, 3 mM dithiothreitol, 500 mM KCL, 30% glycerol).
Ten microliters of the solution was incubated with 90 ml of RT cocktail
(40 mM Tris HCL, pH 7.8, 10 mM MgCl.sub.2, 8 mM dithiothreitol, 94 ml
ddH.sub.2 O, 0.4 U Poly (rA) oligo (dT) [optical density at 260 nm] per ml
and 2.5 mCi/ml .sup.3 H-labeled dTTP) at 37.degree. C. for 1.5 hours. The
reaction mixture was precipitated with 3 ml of 10% trichloroacetic acid
(TCA) and 10 ml of 1% tRNA which served as the carrier, and was then
chilled on ice for 20 minutes. The reaction mixture was filtered through
Whatman GF/C glass microfiber filters and washed 3 times with 5% TCA to
remove unincorporated .sup.3 H-dTTP. Radioactivity was measured in a
liquid scintillation counter.
Single Mutants in gp120
The ability of HXB2-derived mutants (each having one of the 24 N-linked
glycosylation sites mutated by site-directed mutagenesis) to infect
CD4-positive SupT1 cells was compared with that of the wild type virus and
the results are described below. Most of the individual consensus N-linked
glycosylation sites are dispensable for viral infectivity. N-linked
glycosylation sites that are likely to play important roles in HIV-1
infectivity are not randomly distributed in gp120; they are generally
located in the N-terminal half of gp120.
Since deglycosylation of proteins can improve their immunogenicity, a
candidate vaccine for HIV-1 might be a partially glycosylated gp120 with
most of the dispensable N-linked glycosylation sites removed, such that
the conformation of the protein is largely unaltered and the CD4 binding
site is retained.
Each of the 24 potential N-linked glycosylation sites in the gp120 coding
region of the infectious molecular clone HXB2, was individually modified
to generate 24 N-linked glycosylation site mutants (Table 1). In these
mutants, the Asn-X-Ser/Thr attachment sequence was replaced by either
Gln-X-Ser/Thr or His-X-Ser/Thr. The underlying hypothesis was that if a
given N-linked glycosylation site played no significant role in
syncytium-formation or viral infectivity, then such a mutant should retain
its infectivity and its ability to form syncytia. Each of the 24 mutants
was designated by the residue number of the respective N-linked
glycosylation site (Table 1).
Expression of Envelope Proteins
To determine if mutations introduced to any of the 24 N-linked
glycosylation sites affected the expression of the envelope protein, 10
.mu.g each of mutant or wild type proviral DNA was transfected into
3-5.times.10.sup.6 COS-1 cells using DEAE-dextran as described above. Cell
lysates derived from COS-1 transfectants were then examined in western
blots as described above. As shown in FIG. 4, Both gp160 and gp120 were
detected in all 24 mutants, suggesting that no particular individual
N-linked glycosylation site was indispensable for the expression of the
envelope protein.
Syncytium-formation and Viral Infectivity
To evaluate whether mutations introduced into any of the individual
N-linked glycosylation sites affected syncytium-formation and viral
infectivity, cell-free virions obtained from the culture supernatant of
COS-1 transfectants were collected at 48 hours post-transfection. Equal
amounts of mutant and wild type viruses, as measured by RT activity, were
used to infect CD4-positive SupT1 cells. Virus-infected cultures were
monitored for syncytium formation and RT activity. As in the case of the
wild type virus-infected SupT1 cultures, syncytia and RT activity were
detected in all the mutant virus-infected SupT1 cultures (Table 1).
However, 6 mutant viruses, mutants 88, 141, 160, 197, 262 and 276,
exhibited delays in growth kinetics when compared with the wild type virus
(Table 1).
Third-site N-linked Glycosylation Mutants
To examine whether the observed effect on viral infectivity in mutants 88,
141, 160, 197, 262, and 276 was due to amino acid substitutions introduced
to replace the asparagine residue of the canonical N-linked glycosylation
sequence with a non-canonical residue, six third-site N-linked
glycosylation mutants were constructed (Table 2). These six mutants,
designated 90, 143, 162, 199, 264 and 278, are called third-site mutants
because they had the Ser/Thr residue of the Asn-X-Ser/Thr sequence
replaced by a different amino acid residue.
The ability of these six third-site mutants to infect CD4-positive SupT1
cells was also examined. If the phenotype of a third-site N-linked
glycosylation mutant is similar to that of the wild type virus, it is
likely that the observed defect in infectivity for the corresponding
first-site mutant is the result of amino acid substitution at the first
site rather than the loss of that particular N-linked glycosylation site.
For instance, mutant 162 was indeed found to have similar growth kinetics
to the wild type virus (Table 2). This suggested that the impairment of
viral infectivity observed for mutant 160 in SupT1 cells was likely due to
the substitution of asparagine residue with a glutamine residue at this
particular consensus N-linked glycosylation site; but not due to the loss
of this particular consensus N-linked glycosylation site. The remaining
five third-site N-linked glycosylation mutants, like their respective
first-site mutants, all showed partial impairment in infectivity when
compared with the wild type virus (Table 2).
Mutations Introduced at Combinations of N-Linked Glycosylation Sites
Additional mutants in potential N-linked glycosylation sites in gp120 were
generated by oligodeoxynucleotide directed mutagenesis as described above.
The 2.7 Kb SalI-BamHI fragment of the molecular provirus clone HXB2, was
cloned into bacteriophage M13mp18 at SalI-BamHI sites and was used as the
template for mutagenesis. The oligonucleotides used for the mutagenesis
are listed in the Table 1. Changes were made from the consensus N-linked
glycosylation sequence Asn-X-Ser/Thr (N-X-S/T) to either Gln-X-Ser/Thr
(Q-X-S/T) or His-X-Ser/Thr (H-X-S/T). Five mutants were generated each of
which was altered at the amino acids contained within the parentheses as
follows: C2, (386/486); C3(397/463); C4 (386/392/397/463); C5
(386/392/397/406/463); and C6 (386/392/397/406/448/463) (Table 3). The
mutations were confirmed by Sanger sequencing (Sanger et al., 1977, Proc.
Natl. Acad, Sci. USA 74:5463).
Expression of Envelope Proteins and Effect of Combinations of Mutations on
Viral Infectivity
Mutant proviral DNA and wild type DNA (3 .mu.g) was transfected into
3.times.10.sup.6 COS-7 cells (a monkey kidney cell line, CV-1, origin
minus, SV40) using DEAE-dextran as described above. Cell lysates from
COS-7 transfected cells collected 48 hours after transfection were
examined by western blotting. Proteins were separated by
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and
reacted with sheep anti-gp120 antisera (Chou et al., 1988, J. Infect. Dis.
157:805) (FIG. 4). Wild type DNA and all of the C-terminal mutants C2, C3,
C4, C5, and C6 expressed gp160 and gp120 proteins at ratios similar to
each other demonstrating that the position of the mutations has no
apparent effect on cleavage of gp160 to gp120 and gp41. However, the
mobilities of the mutated proteins is higher (faster) than those of the
wild type (FIG. 6, Top) suggesting that some carbohydrates have been
removed from these mutant proteins. In conclusion, oligosaccharides at the
C-terminal region of gp120 appear to be dispensable for cleavage of gp160
to gp120/gp41.
To test the effect of the removal of carbohydrates from the C-terminal
region of gp120 on viral infectivity, cell free virus obtained from these
mutants was used to infect the CD4-positive T cell line, SupT1.
Supernatants were collected from COS-7 transfected cells 48 hours
post-transfection. RT assays were performed and were used as a measure of
the amount of virus in the supernatant. An equal amount of virus, adjusted
to an RT activity of approximately 400K cpm, was used to infect
4.times.10.sup.6 SupT1 cells. The infectivity of wild type and mutant
viruses was determined by examining the cultures for the formation of
syncytia and by measuring RT activity as described above. Syncytia were
apparent in cultures infected with each of the mutants beginning at day 4
postinfection and the formation of syncytia progressed with similar
kinetics in each culture (FIG. 7A). Thus, the carbohydrates at C-terminal
of gp120, which encompass the CD4-binding region, are not essential for
viral infectivity.
To determine whether other regions of gp120 could be deglycosylated without
affecting processing of gp160 and infectivity, another heavily
glycosylated region located at the N-terminus of gp120, from cysteine 126
to cysteine 196, was mutated. The oligonucleotides used for mutagenesis
are summarized in Table 1. Four N-terminal mutants, N2(141/186), N3
(141/160/186), N4 (136/141/160/186), and N5 (136/141/156/160/186) were
generated (Table 3). Mutants N3, N4 and N5 were defective in processing of
gp160. Cultures infected with mutant N2, which had two mutated N-linked
glycosylation sites formed syncytium at day 4 post-infection and had a
higher RT activity than that of the wild type (FIG. 7B). In contrast,
removal of more than three N-linked glycosylation sites (mutants N3, N4,
and N5) in the N-terminal region of gp120 significantly reduced viral
infectivity, in that no syncytia could be observed at any time
postinfection.
The data described above demonstrate that six N-linked glycosylation sites
at the C-terminal of gp120 spanning the CD4-binding region are not
essential for processing of gp160 or for viral infectivity. Binding of
gp120 to CD4 is essential for infection of CD4-positive T cells. The data
described above suggest that carbohydrates that cover the CD4 binding
region are not important for the gp120/CD4 interaction. However,
carbohydrates at the N-terminal Cys 126-196 loop of gp120 are important
for envelope processing and for viral infectivity. For vaccine production,
the N-linked glycosylation sites in the cys 126-196 loop containing the V1
and V2 sequences preferably are to be maintained to provide optimum proper
conformation of the gp120 molecule.
More Detailed Analysis of the Effect of Combinations of Mutants on Viral
Infectivity and Envelope Processing
Using the methods described above, additional combinations of mutations
were introduced into the C-terminal portion of the gp120 of HIV-1 in the
molecular clone HXB2, to study the effect of these mutations on viral
infectivity. The results are presented in Table 4. The amino acid numbers
of the first amino acid in each consensus sequence are listed along the
top of the table. Mutations in any given site are indicated by a "-"
symbol, whereas wild type consensus N-linked glycosylation sites are
indicated by a "+" symbol. It is clear from the data in the table that
some combinations of mutations result in loss of, or impaired infectivity,
while others have no effect. For example, in row S seven N-linked
glycosylation sites have been mutated without affecting viral infectivity
and in row W a combination of eight mutations have been introduced that do
not affect infectivity. In contrast, the particular combination of seven
mutations (shown in rows Q and T) result in impaired infectivity and
additional combinations of nine and ten (see row U and V, respectively)
significantly reduce or eliminate viral infectivity. It is also evident
from the data that the N-linked glycosylation site at amino acid number
289 plays a role in infectivity when other N-linked glycosylation sites in
the C-terminal portion of the molecule are also mutated. Thus, it is
preferable for the mutant protein to have a wild type residue at position
289 if the molecule contains additional C-terminal mutations.
Generation of Partially Deglycosylated gp120 for Use as a Candidate Vaccine
Candidate vaccine gp120 molecules should generally possess the following
properties: 1) they should be partially deglycosylated in the C-terminal
portion of the molecule (defined above) to a sufficient extent to permit
immune recognition of this portion of the molecule; and 2) a sufficient
amount of the wild type conformation of the molecule should be retained
such that the mutant virus substantially retains infectivity. A
recombinant gp120 molecule which satisfies both of these criteria is
likely to elicit a protective immune response to reduce viral infectivity.
Recombinant gp120 molecules derived from any strain of HIV-1 which satisfy
the criteria listed above can be generated using the methods described
above. All that is required is a knowledge of the sequence of the
gp160/gp120 gene in the particular strain of HIV-1 of interest, which if
not already available, can be obtained by a skilled artisan using ordinary
cloning and sequencing technology such as that described in the Molecular
Cloning Manual (Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, NY). Potential N-linked
glycosylation sites can be identified by locating the consensus
Asp-X-Ser/Thr regions and mutations and combinations thereof can be
introduced into these sites as described above. Mutated molecules, wherein
the mutations have substantially no effect on either infectivity, can then
be identified as described above. Such molecules can be obtained by the
skilled artisan without undue experimentation because the techniques and
tests to be used are common and familiar to those knowledgeable in the
art.
In a similar manner to that described above, gp160 molecules can be
generated which are partially glycosylated in the C-terminal portion of
gp120. The methods for generating such molecules are identical to those
described for gp120. Partially deglycosylated gp160 can also be used as a
vaccine candidate provided the C-terminal end of the gp120 portion is
deglycosylated as described.
To determine whether the molecule is sufficiently deglycosylated, its
mobility on a gel be compared to wild type as described above. As
indicated the mutation should produce a gp120 entity of less than 90% of
the wild-type molecular weight. Alternatively, chemical techniques for
quantitating sugar content are well known. See, e.g., Chapin et al. IRL
Press (1986) pp. 178-181 and Methods of Carbohydrate Chemistry Vol. 7
(Whistler et al. Eds.) Academic Press (1976) p. 198 which describe acid
hydrolysis and methanolysis. After methanolic hydrolysis, monosaccharides
are derivatized e.g., to trimethysilyl ethers of the methyl glycosides.
Quantitation is accomplished by gas chromatography using parallel external
standards of monosaccharide mixtures. Alternatively total sugar content of
a glycoprotein of known amino acid sequence can be determined by mass
spectroscopy to obtain accurate mass of glycosylated and unglycosylated
moieties.
Expression of Recombinant Partially Deglycosylated gp120
Large quantities of recombinant partially deglycosylated gp120 or gp160
mutant glycoproteins can be obtained by expressing these proteins in a
number of expression systems. For example, chinese hamster ovary (CHO)
cells can be transfected with a plasmid encoding a mutated gp120 or gp160
gene, using any number of transfection methods all of which are described
in detail in Sambrook et al. (Supra). Mutated proteins can be expressed in
a constitutive manner under the control of its own promoter under the
control of another promoter such as another retrovirus LTR. Alternatively,
mutated proteins can be expressed in an inducible manner, wherein
expression is driven by a promoter that responds to the addition of an
inducer molecule to the transfected cells. Examples of such promoters can
be found in Sambrook et al. (Supra). Glycoproteins that are so expressed
can be recovered from the cells and from the cell medium using common
biochemical techniques. See Lasky et al. Science 233:209-212 (1986); Robey
et al. Proc. Nat'l. Acad. Sci. 83:7023-7027 (1986); Pyle et al. Aids
Research and Human Retrovirus 3:387-399 (1987).
A baculovirus expression system can also be used to obtain large quantities
of partially glycosylated gp120 or gp160. A gene encoding a mutated
glycoprotein can be cloned into a commercially available baculovirus
transfer plasmid. A recombinant baculovirus encoding such a protein can be
generated as described by Summers and Smith (1988, A Manual of Methods for
Baculovirus Vectors and Insect Cell Culture Procedures: Texas Agricultural
Experiment Station Bulletin No. 1555, College Station, Tex.). The virus
can be used to infect insect cells, such as Sf9 cells, whereupon the
mutated glycoprotein will be expressed to high levels as the baculovirus
replicates. Protein is recovered from the culture using ordinary standard
biochemical techniques.
The mutated proteins can also be produced as part of a viral particle, with
or without alterations to other portions of the virus. See, e.g. the
method of Aldovini et al. J. Virol. 64:1920-1926 (1990).
Generation of Antibodies
Recombinant envelope proteins can be used to generate antibodies using
standard techniques, well known to those in the field. For example, the
proteins are administered to challenge a mammal such as a goat, rabbit or
mouse. The resulting antibodies can be collected as polyclonal sera, or
antibody-producing cells from the challenged animal can be immortalized
(e.g. by fusion with an immortalizing fusion partner) to produce
monoclonal antibodies. Monoclonal antibody-producing hybridomas (or
polyclonal sera) can be screened for antibody binding to the protein and
to wild type envelope. They can also be screened for the ability to
neutralize infectivity of HIV-1 isolates, preferably multiple (e.g., at
least 3) isolates each having diverse sequences in the hypervariable V3
region. By antibodies we include constructions using the binding
(variable) region of such antibodies, and other antibody modifications.
Vaccines
The mutant envelope protein may be formulated into vaccines according to
standard procedures known to those in the field. For example, procedures
currently used to make wild-type envelope protein vaccines (e.g.,
Microgenysys gp160 vaccine) can be used to make vaccines with the
selectively deglycosylated envelope protein. Various modifications such as
adjuvants and other viral or toxin components known for such vaccines or
immunotherapeutics may be incorporated with the mutants.
TABLE 1
__________________________________________________________________________
N-linked Glycosylation Mutants of HXB2 Envelope Glycoprotein
__________________________________________________________________________
MUTANT
AMINO ACID
MUTAGENIC
VIRUS
CHANGE OLIGONUCLEOTIDE(5' to 3')* VIRAL INFECTIVITY
__________________________________________________________________________
88 Asn to Gln
TAGTATTGGTACAGGTGACAGAAAATTT
(SEQ ID NO:1)
+**
136 Asn to Gln
TGATTTGAAGCAGGATACTAATAC
(SEQ ID NO:2)
+
141 Asn to Gln
ATACTAATACCCAAAGTAGTAGCGGGA
(SEQ ID NO:3)
+**
156 Asn to Gln
GATAAACAGTGCTCTTTCAATAT
(SEQ ID NO:4)
+
160 Asn to Gln
CTGCTCTTTCCAGATCAGCACAAG
(SEQ ID NO:5)
+**
186 Asn to Gln
TACCAATAGATCAGGATACTACCAGC
(SEQ ID NO:6)
+
197 Asn to Gln
TGACAAGTTGTCAGACCTCAGTCAT
(SEQ ID NO:7)
+**
230 Asn to His
TAAAATGTAATCATAAGACGTTCA
+ (SEQ ID NO:8)
234 Asn to His
ATAAGACGTTCCATGGAACAGGACCA
+ (SEQ ID NO:9)
241 Asn to Gln
GACCATGTACACAGGTCAGCACAGTAC
(SEQ ID NO:10)
+
262 Asn to Gln
ACTGCTGTTACAAGGCAGTCTAG
(SEQ ID NO:11)
+**
276 Asn to Gln
TTAGATCTGTCCAGTTCACGGACAAT
(SEQ ID NO:12)
+**
289 Asn to Gln
TAGTACAGCTGCAGACATCTGTAGAAA
(SEQ ID NO:13)
+
295 Asn to Gln
CTGTAGAAATTCAATGTACAAGAC
(SEQ ID NO:14)
+
301 Asn to His
ACAAGACCCAACCACAATACAAGAAA
+ (SEQ ID NO:15)
332 Asn to His
GCACATTGTCACATTAGTAGAGC
+ (SEQ ID NO:16)
339 Asn to Gln
GCAAAATGGCAGAACACTTTAAAAC
(SEQ ID NO:17)
+
356 Asn to Gln
ATTCGGAAATCAGAAAACAATAATCTTTA
(SEQ ID NO:18)
+
386 Asn to Gln
TTTCTACTGTCAGTCAACACAACTG
(SEQ ID NO:19)
+
392 Asn to Gln
ACAACTGTTTCAGAGTACTTGGTTTAATAG
(SEQ ID NO:20)
+
397 Asn to Gln
GTACTTGGTTTCAGAGTACTTGGAG
(SEQ ID NO:21)
+
406 Asn to His
CTGAAGGGTCACATAACACTGAAGGA
+ (SEQ ID NO:22)
448 Asn to Gln
GATGTTCATCACAGATTACAGGGCTG
(SEQ ID NO:23)
+
463 Asn to His
GGTAATAGCAACCATGAGTCCGAGAT
+ (SEQ ID NO:24)
__________________________________________________________________________
* Underlined type indicates mutation sites,
**Partial impairment
TABLE 2
__________________________________________________________________________
Third-site N-linked Glycosylation Mutants of HXB2 Envelope
__________________________________________________________________________
Glycoprotein
MUTANT
AMINO ACID
MUTAGENIC
VIRUS
CHANGE OLIGONUCLEOTIDE(5' to 3')* VIRAL INFECTIVITY
__________________________________________________________________________
90 Thr to Val
GGTAAATGTGGTCGACAACTTTTGACATGT
(SEQ ID NO:25)
+**
143 Ser to Ala
AATACCAATAGTGCATGCGGGAGAATGG
(SEQ ID NO:26)
+**
162 Ser to Ala
CTGCTCTTTCAATATTGCCACAAGCATAAG
(SEQ ID NO:27)
+
199 Thr to Glu
GTTGTAACACCGAAGTCATTACACAG
(SEQ ID NO:28)
+**
264 Ser to Ala
CTGCTGTTAAATGGCGCTCTAGCAGAAGAAGAG
(SEQ ID NO:29)
+**
278 Thr to Val
CTGTCAATTTCGTCGTCGACAATGCTAAA
(SEQ ID NO:30)
+**
__________________________________________________________________________
* Underlined type indicates mutation sites
** Partial impairment
TABLE 3
______________________________________
Combination N-linked glycosylation sites mutants of
HXB2 env glycoprotei
gp160 viral
Mutant amino acid change
cleavage infectivity
______________________________________
C2 386/463 + +
C3 397/463 + +
C4 386/397/406/463
+ +
C5 386/392/397/406/463
+ +
C6 386/392/397/406/448/463
+ +
N2 141/186 + +
N3 141/160/186 +* -
N4 141/156/160/186
- -
N5 141/136/156/160/186
- -
______________________________________
+*: severe impairment
TABLE 4
__________________________________________________________________________
Additional combinations of N-Linked Glycosylation Mutants in the
C-Terminal Portion of gp120
N-Linked Glycosylation Site Amino Acid Number
Viral
Row #
230
234
289
301
332
339
356
386
392
397
406
448
463
Infectivity
__________________________________________________________________________
Q + + + + - - - - + - - + - +
R + + + + - - + - + - - + - ++
S + + + - - - + - + - - + - ++
T + + - + - - + - + - - + - +
U - - - + - - + - + - - + - -
V - - - - - - + - + - - + - -
W - - + + - - + - + - - + - ++
__________________________________________________________________________
__________________________________________________________________________
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# single) STRANDEDNESS:
# linearOPOLOGY:
#28: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 26 CATT ACACAG
- (2) INFORMATION FOR SEQ ID NO: 29:
- (i) SEQUENCE CHARACTERISTICS:
# 33) LENGTH:
# nucleic acid
# single) STRANDEDNESS:
# linearOPOLOGY:
#29: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 33 CTCT AGCAGAAGAA GAG
- (2) INFORMATION FOR SEQ ID NO: 30:
- (i) SEQUENCE CHARACTERISTICS:
# 29) LENGTH:
# nucleic acid
# single) STRANDEDNESS:
# linearOPOLOGY:
#30: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 29 CGAC AATGCTAAA
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